Alcohol dehydrogenase (ADH) of Drosophila not only catalyzes the oxidation of ethanol to acetaldehyde, but additionally catalyzes the conversion of this highly toxic product into acetate. This mechanism is demonstrated by using three different methods. After electrophoresis the oxidation of acetaldehyde is shown in an NAD-dependent reaction revealing bands coinciding with the bands likewise produced by a conventional ADH staining procedure. In spectrophotometric measurements acetaldehyde is oxidized in an NADdependent reaction. This activity is effectively inhibited by pyrazole, a specific inhibitor of ADH. By means of gas chromatographic analysis a quick generation of acetate from ethanol could be demonstrated. Our conclusion is further supported by experimental results obtained with either purified ADH F enzyme or genotypes with or without ADH, aldehyde-oxidase, pyridoxal-oxidase and xanthine-dehydrogenase activity. These results are discussed in relation to ethanol tolerance in the living organism in particular with respect to differences found between ADH in Drosophila melanogaster and D. simulans, and in relation to the possible implications for the selective forces acting on ADH-polymorphism.

Until recently the alcohol dehydrogenase of Drosophila melanogaster was thought to act only in the first step of primary alcohol oxidation, producing an aldehyde. Instead, acetic acid is the main product of a two-step process. A rapid procedure was developed for the isolation and purification of two allozymes. The thermostability of the purified enzymes was found to be very different, t 1/2 at 35 degrees C, being 45 min and 130 min for ADH-F and ADH-71k respectively. The kinetic parameters of ethanol oxidation by the two purified allozymes were determined within physiological substrate and coenzyme ranges. The use of artificial electron acceptors has a notable influence on the ethanol oxidation: the apparent Michaelis constants increase; the oxidation rate with ADH-71k increases, whereas it decreases with ADH-F. Purified ADH is shown to be able to catalyze the oxidation of acetaldehyde solely in the presence of NAD+, and PMS and MTT as artificial electron acceptors. From the kinetic data the relative in vivo oxidation rates of ethanol by both ADH allozymes were calculated. ADH-F turned out to be somewhat less effective (30%-40%) than ADH-71k. The physiological consequences of these differences are discussed.

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Drosophila melanogaster strains, homozygous for the alcohol dehydrogenase alleles AdhF, AdhS, and Adhn4 respectively, were tested for oviposition site preference with a Multiple Choice Device consisting of 18 patches per choice disk. Equal numbers of patches with ethanol-, acetic acid-, and water-supplemented medium were offered simultaneously. Patches with acetic acid-supplemented medium were chosen predominantly as oviposition sites. Pretreatment of flies with increasing concentrations of 2-propanol to inhibit alcohol dehydrogenase (ADH) activity resulted not only in a decreasing choice of acetic acid patches, but also in the laying of a decreasing number of eggs. Adh-null mutant flies showed a similar change in behavior pattern after 2-propanol treatment. Therefore it was concluded that ADH activity is not involved primarily in oviposition site preference behavior. A complicating factor is acetone, the oxidation product of 2-propanol, which had an even larger impact on egg production. However, differences in ADH allozymes with respect to biochemical oxidation capacity of secondary alcohols will not necessarily lead to differences between the Adh genotypes in oviposition rates or apparent changes in preferences, due to additional biochemical differences in inhibition rates by acetone of the various allozymes and other enzyme systems.